Fluorescent labeling of oligonucleotides is a technique commonly used in structural and functional studies of oligonucleotides. A wide range of fluorescent reporter groups have been in use, many of these are commercially available (The Handbook, A guide to fluorescent probes and labeling technologies, 10th edition; Haugland, Richard P.; Invitrogen Corp., 2005, pp 315-412). Among these fluorophores, fluorescein, rhodamine and cyanine are the most commonly used. These fluorophores cover a wide range of excitation and emission wavelengths and are in general highly fluorescent; however, most of these fluorophores are charged molecules and the fluorescent properties are often pH and environment dependent. In addition, many of these fluorescent probes are not photostable. The cyanine derivatives Cy3 and Cy5 are the only red-purple emitting fluorophores that are currently available for oligonucleotide labeling through the phosphoramidite chemistry, however, their applications are limited by their (photo)chemical instability. There exists a high demand for such red-purple fluorophores.
Numerous 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY™)
derivatives have been known to possess very high, and some near unity, fluorescent quantum yields that are relatively insensitive to changes in pH and polarity and are stable under many biological conditions (Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891; Ulrich, G.; Ziessel, R.; Harriman, A. Angew. Chem., Int. Ed. 2008; 47, 1184). These fluorophores are often more photostable than many fluorescein, rhodamine and cyanine analogues. Therefore the BODIPY™ fluorophores have found useful applications in nucleic acid labeling. To date, BODIPY™ labeling of oligonucleotides has been carried out post-synthetically between an amino modifier on oligonucleotides and an activated BODIPY™ carboxylate ester (The Handbook, A guide to fluorescent probes and labeling technologies, 10th edition; Haugland, Richard P.; Invitrogen Corp., 2005, pp 315-412).
U.S. Pat. No. 5,861,287 describes methods for the use of BODIPY™ fluorophore-labelled DNA for dye-primer sequencing. This patent describes a BODIPY™ phosphoramidite derivative where the phosphoramidite is attached at the 3-position of the indacene ring skeleton. Canadian patent application number 2,577,053 describes several fluorescent dyes, including BODIPY™ dyes, substituted by at least one phosphonate group. The only phosphoramidite compounds specifically mentioned contained the phosphoramidite at the 4-position of the indacene ring skeleton. Neither of these documents reports the use of these phosphoramidite BODIPY™ compounds in solid phase oligonucleotide synthesis.